Method For Producing 3-Hydroxypropionaldehyde

ABSTRACT

A method for producing 3-hydroxypropionaldehyde from glycerin in high conversion ratio is provided. The method is characterized by comprising a step of dehydrating glycerin using a microbial cell and/or a treated microbial cell containing diol dehydratase and/or glycerol dehydratase, and optionally diol dehydratase reactivating factor and/or glycerol dehydratase reactivating factor, under conditions so as to give a value (X/Y 2 ) calculated by dividing a catalytic amount [X (U/g glycerin)] of diol dehydratase and/or glycerol dehydratase by square of glycerin concentration [Y (g/100 ml)] within a range of 10 to 8,000, to produce 3-hydroxypropionaldehyde.

TECHNICAL FIELD

The present invention relates to a method for producing3-hydroxypropionaldehyde and a method for producing 1,3-propanediol,3-hydroxypropionic acid, acrolein, acrylic acid and an acrylic esterproduced from 3-hydroxypropionaldehyde produced by the method. Inparticular, the present invention relates to a method which canefficiently produce 3-hydroxypropionaldehyde in high purity and a methodwhich can efficiently produce 1,3-propanediol, 3-hydroxypropionic acid,acrolein, acrylic acid and an acrylic ester in high purity from the3-hydroxypropionaldehyde.

BACKGROUND ART

1,3-Propanediol is a useful compound having a wide range ofapplications, for example, as a monomer used for producing polyester andpolyurethane, and a starting material for synthesizing a cycliccompound.

As a method for synthesizing 1,3-propanediol, both of a method bychemical synthesis and a method by fermentation have been well-known. Asthe former method, for example, a method for producing 1,3-propanediolvia carbonylation of ethylene oxide using a rhodium catalyst (forexample, U.S. Pat. No. 4,873,378, U.S. Pat. No. 873,379 and U.S. Pat.No. 4,935,554), and a method for producing 1,3-propanediol by reducing3-hydroxypropionaldehyde (for example, U.S. Pat. No. 2,434,110) havebeen known.

However, the method by the chemical synthesis is not sufficient inconversion ratio and selectivity, and not favorable in the viewpoint ofcost, because a purification process is required to remove a by-product.In addition, if 3-hydroxypropionaldehyde as a raw material contains aby-product, a secondary product might be further formed in thesubsequent production process of 1,3-propanediol, and this could requirea difficult purification, or cause discoloration or undesirablepolymerization in products such as fibers in a production of textilesusing this 1,3-propanediol as a raw material thereafter. For thisreason, a content of by-product in 3-hydroxyprpionaldehyde to be used asa raw material for producing 1,3-propanediol is desirably as low aspossible.

Further, the latter method is a method of producing 1,3-propanediolthrough the fermentation of glycerin or glucose using a strain producing1,3-propanediol such as Citrobacter, Clostridium, Enterobacter,Ilyobacter, Klebsiella, Lactobacillus and Pelobacter, and the like. Thismethod comprises two-step reaction consisting of a step of convertingglycerin to 3-hydroxypropionaldehyde (3-HPA) and water with adehydratase and a step of reducing the resultant 3-HPA to1,3-propanediol with a NAD⁺-link-oxidoreductase. In addition, to improveyield of desired 1,3-propanediol, a method for producing 1,3-propanediolfrom glycerin using a recombinant microorganism has been disclosed (forexample, WO 98/21339, WO 99/58686, U.S. Pat. No. 6,025,184 and WO01/12833).

However, in the production method by the fermentation, it has been knownthat, to obtain NADH necessary for the latter reaction among thereactions, the two-step reaction and a reaction to form NADH bydehydrogenation to dihydroxyacetone occur simultaneously. By thisreason, there is a problem that a conversion ratio from glycerin to1,3-propanediol by the general fermentation becomes as low as around 50%resulting in an insufficient yield of 3-HPA. To solve this problem,production of 1,3-propanediol using a recombinant microorganism has beenreported. However, even if such microorganism is used, since a reactionto form NADH needs to occur in addition to the reaction from glycerin to3-HPA by the similar reason to the above, it is very difficult to attaina high conversion ratio. Further, a fermentation culture generallycontains many by-products such as nutrients contained in the culture andmicrobial products. For this reason, the method using fermentation isalso not preferable from an economical viewpoint, because a purificationprocess becomes complicated much more than that by chemical synthesis.In addition to the problem, there is another problem that the method bythe fermentation often uses an organic solvent such as cyclohexane in apurification process of the desired product, 1,3-propanediol, andfurther needs post-treatment of such organic solvent considering theenvironment.

On the other hand, 3-hydroxypropionaldehyde, which is an intermediate inthe method, can be used also as an intermediate in the production of3-hydroxypropionic acid, acrolein, acrylic acid and acrylic esters, aswell as 1,3-propanediol as described above. Among these products,acrylic acid, for example, has a wide range of applications, and hasbeen used as a copolymer for acrylic fiber, adhesives or agglutinant inemulsion, as well as coating materials, textile processing, leather,construction materials, and the like. Production method for acrylic acidhas been conventionally known, and it is generally produced by atwo-step gas phase catalytic oxidation, that is, from propylene toacrolein and from acrolein to acrylic acid. In this case, acrolein usedas an intermediate material can be also produced via treatment of3-hydroxypropionaldehyde under acidic conditions. Accordingly, toproduce acrylic acid in high purity and at low cost, it is desirablethat acrolein and further 3-hydroxypropionaldehyde are efficientlyproduced in high purity.

As a method for producing 3-hydroxypropionaldehyde (3-HPA), a method forproducing 3-HPA from glycerol using a recombinant strain obtained bycloning of diol dehydratase and/or glycerol dehydratase in combinationwith diol dehydratase reactivating factor and/or glycerol dehydratasereactivating factor (for example, Journal of Bacteriology, Vol. 181, No.13, pp. 4110-4113, 1999; The Journal of Biological Chemistry, Vol. 272,No. 51, pp. 32034-32041, 1997; Arch. Microbiol., 174:81-88 (2000); TheJournal of Biological Chemistry, Vol. 274, No. 6, pp. 3372-3377, 1999),and a method for converting glycerin to 3-HPA by culturing Klebsiellapneumoniae in a glycerin-rich culture medium followed by suspending in abuffer containing semicarbazide and glycerin (Applied and EnvironmentalMicrobiology, Vol. 50, No. 6, pp. 1444-1450, 1985) have been reported.Among the methods, however, the method for producing 3-HPA from glycerinusing a recombinant strain is, in any case, a method to scholasticallystudy a behavior in an initial stage of the reaction, and specificreaction conditions of the conversion from the glycerin to 3-HPA needmore study from the industrial viewpoint. Further, in the latter method,though a yield of around 83% at most can be attained due to anaccumulation of 3-HPA facilitated by the presence of a semicarbazide,when 3-HPA is recovered in the presence of the semicarbazide, 3-HPAforms a complex with the semicarbazide as shown in the followingreaction scheme, from which 3-HPA cannot be recovered again. Thus, themethod cannot provide 3-HPA as a simple substance.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Thus, an object of the present invention is to provide specificconditions for producing 3-hydroxypropionaldehyde (3-HPA) inindustrially high conversion ratio (that is, in high yield).

Another object of the present invention is to provide a method forproducing 1,3-propanediol from the 3-HPA efficiently.

Further another object of the present invention is to provide a methodfor producing 3-hydroxypropionaldehyde acid from the 3-HPA efficiently.

Still further another object of the present invention is to provide amethod for producing acrolein, acrylic acid or an acrylic ester from the3-HPA in high purity and in high yield.

Means to Solve the Problems

It has been generally well-known that activity (amount) of enzyme to actas a catalyst and concentration of substrate greatly influenceconversion ratio. The present inventors have, after extensively studyinga method for converting glycerin to 3-HPA to attain the objects inconsideration of the circumstances, obtained such knowledge that thereis a very strong correlation between catalytic activity and substrateconcentration, in particular, in the conversion. Further, based on thisknowledge, the present inventors have, after extensively studying aconversion from glycerin to 3-HPA using a microbial cell,toluene-treated microbial cell or immobilized microbial cell having dioldehydratase and/or glycerol dehydratase (herein collectively referred toas “diol/glycerol dehydratase” or simply as “dehydratase”) and dioldehydratase reactivating factor and/or glycerol dehydratase reactivatingfactor (herein collectively referred to as “diol/glycerol dehydratasereactivating factor” or simply as “dehydratase reactivating factor”),found that by applying an action of dehydratase with an amount ofcatalyst controlled so as to give a value (X/Y²) calculated by dividinga catalytic amount [X (U/g glycerin)] by square of glycerinconcentration [Y (g/100 ml)] within a specified range, a conversionratio in a high level corresponding to the industrial level, inparticular, a conversion ratio not less than 70% can be attained, and3-HPA can be produced in a high yield. Still further, the presentinventors have also found that since almost all components other thanglycerin such as nutrients are not used, particularly when a treatedmicrobial cell is used, 3-hydroxypropionaldehyde can be easily producedin high purity only by removing the treated microbial cells byfiltration or the like. Furthermore, the present inventors have obtainedsuch knowledge that by hydrogenation of the thus obtained3-hydroxypropionaldehyde, desired 1,3-propanediol can be produced inhigh purity and in high yield.

Further, the present inventors have also obtained such knowledge thatacrolein can be obtained by reacting 3-hydroxypropionaldehyde scarcelycontaining by-products obtained as described above under acidicconditions, and this acrolein can be produced in high purity because thestarting material scarcely contains any by-product, hence, from thisacrolein, acrylic acid can be produced in high purity. In addition, thepresent inventors found that an acrylic ester can be also produced inhigh purity simply by the oxidative esterification of acrolein.

Namely, the object can be attained by a method for producing3-hydroxypropionaldehyde which comprises a step of dehydrating glycerinusing a microbial cell and/or a treated microbial cell containing dioldehydratase and/or glycerol dehydratase, and optionally diol dehydratasereactivating factor and/or glycerol dehydratase reactivating factor,under conditions so as to give a value (X/Y²) calculated by dividing acatalytic amount [X (U/g glycerin)] of diol dehydratase and/or glyceroldehydratase by square of glycerin concentration [Y (g/100 ml)] within arange of 10 to 8,000, to produce 3-hydroxypropionaldehyde.

The another object can be attained by a method for producing1,3-propanediol comprising a step of hydrogenating3-hydroxypropionaldehyde produced by the method to produce1,3-propanediol.

The further another object can be attained by a method for producing3-hydroxypropionic acid comprising a step of oxidizing3-hydroxypropionaldehyde produced by the method to produce3-hydroxypropionic acid.

The still further another object can be attained by a method forproducing acrolein comprising a step of removing microbial cell and/ortreated microbial cell from the reaction product containing3-hydroxypropionaldehyde produced by the method, followed by reacting3-hydroxypropionaldehyde under acidic conditions to produce acrolein; amethod for producing acrylic acid comprising a step of oxidizingacrolein produced by the method to produce acrylic acid; and a methodfor producing an acrylic ester comprising a step of subjecting acroleinproduced by the method to oxidative esterification to produce acrylicester.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail below.

A first aspect of the present invention relates to a method forproducing 3-hydroxypropionaldehyde which comprises a step of dehydratingglycerin using a microbial cell and/or a treated microbial cellcontaining diol dehydratase and/or glycerol dehydratase, and optionallydiol dehydratase reactivating factor and/or glycerol dehydratasereactivating factor, under conditions so as to give a value (X/Y²)calculated by dividing a catalytic amount [X (U/g glycerin)] of dioldehydratase and/or glycerol dehydratase by square of glycerinconcentration [Y (g/100 ml)] within a range of 10 to 8,000, to produce3-hydroxypropionaldehyde. This means that among important factorsconsidered to affect on a conversion ratio from glycerin to 3-HPA, inparticular, an amount of catalyst greatly affects on a conversion ratio,and it has been revealed that by acting dehydratase on glycerin in suchan amount that a value (X/Y²) calculated by dividing a catalytic amount[X (U/g glycerin)] of diol dehydratase and/or glycerol dehydratase bysquare of glycerin concentration [Y (g/100 ml)] is in a specified rangeas of 10 to 8,000, glycerin is selectively utilized in a conversion to3-HPA without an occurrence of undesirable reaction such as a conversionof glycerin to NADH, and glycerin can be converted to 3-HPA in such ahigh conversion ratio as not less than 80%. Further, since conversionfrom glycerin to 3-HPA occurs preferentially and3-hydroxypropionaldehyde to be produced scarcely contains a by-product,3-hydroxypropionaldehyde can be produced in high purity by such a simpleprocedure as to separate/remove microbial cell/treated microbial cell.

In the present invention, a value (X/Y²) calculated by dividing acatalytic amount [X (U/g glycerin)] of diol dehydratase and/or glyceroldehydratase by square of glycerin concentration [Y (g/100 ml)] is in therange of 10 to 8,000. In this case, when X/Y² is fallen in this range, ahigh conversion ratio, for example, a conversion ratio of not less than70% can be attained. On the contrary, when the X/Y² deviates from therange, the conversion ratio from glycerin to 3-HPA remarkably decreases.Lower limit of the X/Y² is preferably 7,000, 6,500, 5,500 and 5,000 inthis order.

As used herein, “glycerin concentration [Y (g/100 ml)]” represents aweight of glycerin contained in 100 ml of a solution. For example, since1 L of 50 mM potassium phosphate buffer (pH 8) containing 1 M ofglycerin contains 92 g of glycerin, a glycerin concentration (Y) of thiscase becomes 9.2 (g/100 ml).

Further, as used herein, “catalytic amount [X (U/g glycerin)]”represents a value of a unit (U) of diol/glycerol dehydratase enzymedivided by a weight (g) of glycerin. In this case, “1 U” as a unit ofenzyme means an ability of microbial cell and/or treated microbial cellexhibiting an activity of diol/glycerol dehydratase enzyme to convert 1μmol of glycerin to 3-HPA per 1 minute, corresponds to an ability ofmicrobial cell and/or treated microbial cell exhibiting a diol/glyceroldehydratase enzyme activity to convert 1 μmol of 1,2-propanediol topropionaldehyde per 1 minute. In this connection, in the presentinvention, the formations of propionaldehyde and3-hydroxypropionaldehyde were detected by a detection method using3-methyl-2-benzothiazolinonehydrazone hydrochloride. For example, when atoluene-treated microbial cell having an activity of 200 U is added to 1L of 50 mM potassium phosphate buffer (pH 8) containing 1 M of glycerin,a catalytic amount (X) becomes 2.17 (U/g glycerin) (=200/92).

In the present invention, glycerin concentration (Y) is not especiallylimited, and may be selected, as appropriate, depending on viscosity ofreaction liquid, amount of enzyme to be added, kind and intensity ofenzymatic activity, concentration and purification processes afterreaction. Lower limit of glycerin concentration (Y) is preferably notless than 0.5 g, more preferably not less than 1 g, and most preferablynot less than 2 g, per 100 ml of solution. Also, upper limit of glycerinconcentration (Y) is preferably not more than 60 g, more preferably notmore than 50 g, and most preferably not more than 40 g, per 100 ml ofsolution. In this case, when glycerin concentration is less than 0.5g/100 ml, due to too low glycerin amount in a solution, an amount of3-HPA to be formed would become unduly low, and the concentration couldbe not economical from the industrial viewpoint. On the contrary, whenglycerin concentration is over 60 g/100 ml, a viscosity of reactionliquid would increase thereby making homogeneous mixing with microbialcell/treated microbial cell difficult, glycerin could be impossible toefficiently receive an action of diol dehydratase/glycerol dehydratase.

In the present invention, dehydratase including diol dehydratase andglycerol dehydratase is coenzyme B12 dependent type as described above,and presence of coenzyme B12 is inevitable to convert glycerin to 3-HPA.Amount of coenzyme B12 to be present in the conversion from glycerin to3-HPA is not especially limited so long as the conversion from glycerinto 3-HPA proceeds sufficiently thereby, and differs depending onconcentration of substrate and the like. An amount of coenzyme B12 to bepresent is preferably in the range of 1 to 1,000 μM, more preferably 10to 800 μM in a concentration of coenzyme B12 per 50 mM of substrateconcentration. Further, amount of microbial cell and/or treatedmicrobial cell is not especially limited so long as the catalytic amountof enzyme satisfies the relation: X/Y²=10 to 8,000, and a conversion ofglycerin to 3-HPA sufficiently proceeds therewith, and differs dependingon form of microbial cell and/or treated microbial cell to be used(microbial cell, immobilized microbial cell and immobilized factor) andsource thereof, concentration of substrate (glycerin) and volume ofreaction liquid. For example, microbial cell and/or treated microbialcell may be used in a batch type or in a flow reaction. When immobilizedmicrobial cell is used in a flow reaction, an amount of microbial celland/or treated microbial cell differs depending on kind of microorganismto be used, life of enzyme, flow rate of reaction liquid (LHSV) and thelike. Also, an amount of microbial cell and/or treated microbial cell isnot especially limited so long as the conversion of glycerin to 3-HPAsufficiently proceeds therewith. It usually about 10 to 100 times ofamount as compared to that of batch type is used. Lower limit ofcatalytic amount (X) of enzyme, although be easily calculated from thevalue of X/Y², is preferably not less than 2.5 U/g glycerin, forexample. Similarly, upper limit of catalytic amount (X) of enzyme can bealso easily calculated from the value of X/Y², suitable value of Y andthe like, but it is preferably not more than 27,000,000 U. In this case,when the catalytic amount of enzyme is less than 2.5 U/g glycerin, anaction of enzyme to glycerin could be insufficient, and yield of desired3-HPA could be also insufficient. On the contrary, even when a catalyticamount is over 28,800,000 U/g glycerin, effects obtained by amount ofenzyme added could fail to be obtained which is not preferable from theeconomical viewpoint. In this connection, as for unit of the enzyme, anability of microbial cell and/or treated microbial cell exhibitingdiol/glycerol dehydratase enzymatic activity to convert 1 μmol of1,2-propanediol to propionaldehyde is defined as “1 U”, and measuringmethod thereof is not especially limited, and any method can be used solong as formation of propionaldehyde from 1,2-propanediol can bedetected thereby. In the present invention, formations ofpropionaldehyde and 3-hydroxypropionaldehyde were detected by a methodto detect using 3-methyl-2-benzothiazolinone hydrochloride.

In the present invention, “diol/glycerol dehydratase” or “dehydratase”is an enzyme having a catalytic action to convert glycerin to3-hydroxypropionaldehyde (herein also referred to as “3-HPA”) and waterby dehydration. Any known enzyme can be used without special limitationso long as the enzyme has such an action. Among these enzymes, in viewof life of enzyme, glycerol dehydratase is preferably used.

In the present invention, glycerol dehydratase is not especiallylimited, and glycerol dehydratase derived from any sourcehaving/expressing this enzyme may be used. Specifically, these includemicroorganisms belonging to Klebsiella genus, Citrobacter genus,Clostridium genus, Lactobacillus genus, Enterobacter genus, Caloramatorgenus, Salmonella genus and Listeria genus, and more specifically,glycerol dehydratase derived from Klebsiella pneumoniae, Citrobacterpneumoniae, Clostridium Pasteurianum, Lactobacillus leichmannii,Citrobacter intermedium, Lactobacillus reuteri, Lactobacillus buchneri,Lactobacillus brevis, Enterobacter agglomerans, Clostridium butyricum,Caloramator viterbensis, Lactobacillus collinoides, Lactobacillushilgardii, Salmonella typhimurium, Listeria monocytogenes and Listeriainnocua. These glycerol dehydratases may be used alone or in combinationof two or more kinds, or may be used in combination with dioldehydratase described in detail below.

Method for isolating glycerol dehydratase from the sources is notespecially limited, and glycerol dehydratase may be isolated from anymicroorganism as described above similarly to a known separation andisolation method such as extraction and column chromatography.

Further, in the present invention, diol dehydratase is also notespecially limited. Any diol dehydratase derived from any sourcehaving/expressing this enzyme may be used. Specifically, these includemicroorganisms belonging to Klebsiella genus, Propionibacterium genus,Clostridium genus, Lactobacillus genus, Salmonella genus, Citrobactergenus, Flavobacterium genus, Acetobacterium genus, Brucella genus andFusobacterium genus, and more specifically, diol dehydratase derivedfrom Klebsiella pneumoniae, Propionibacterium freudenreichii,Clostridium glycolicum, Lactobacillus brevis, Salmonella typhimurium,Citrobacter freundii, Lactobacillus buchneri, Brucella melitensis,Fusobacterium nucleatum, Klebsiella oxytoca, Salmonella typhimurium,Listeria monocytogenes and Listeria innocua. These diol dehydratases maybe used alone or in combination of two or more kinds, or may be used incombination with glycerol dehydratase described in detail above.

Method for isolating diol dehydratase from the sources is not especiallylimited, and diol dehydratase may be isolated from any microorganism asdescribed above similarly to a known separation and isolation method ofenzyme such as extraction and column chromatography.

Microbial cell and/or treated microbial cell may contain, in addition todiol/glycerol dehydratase, diol/glycerol dehydratase reactivatingfactor. In this case, in view of conversion ratio of glycerin to 3-HPAand reactivation of enzyme, microbial cell/treated microbial cell havingdiol/glycerol dehydratase reactivating factor is preferably used.

In the present invention, “diol/glycerol dehydratase reactivatingfactor” or “dehydratase reactivating factor” is referred to as a factorto induce again (reactivate) an activity of dehydratase, which has beendeactivated by catalyzing a reaction of glycerin to 3-HPA+H₂O. In moredetail, the coenzyme B12 is involved in the reaction of glycerin to3-HPA+H₂O, which is catalyzed by dehydratase, and active center of thedehydratase is deactivated after developing and catalyzing the reactionof glycerin to 3-HPA+H₂O due to collapse of the coenzyme B12. Here, thedehydratase reactivating factor replaces the collapsed coenzyme B12 witha new coenzyme B12 to induce again (reactivate) an activity of thedehydratase and make the dehydratase reusable for dehydration reactionof glycerin. Thus, in the present invention, a factor having an abilityto reactivate the dehydratase is referred to as “diol/glyceroldehydratase reactivating factor” or “dehydratase reactivating factor”.The dehydratase reactivating factor is not especially limited so long asthe factor has an ability as described above, and well-known glyceroldehydratase reactivating factor which activates deactivated glyceroldehydratase and diol dehydratase reactivating factor which activates adeactivated diol dehydratase can be used. In this connection,dehydratase and dehydratase reactivating factor may be used in anycombination of those described above. Dehydratase reactivating factor ispreferably composed of a large subunit of diol dehydratase reactivatingfactor and/or glycerol dehydratase reactivating factor and a smallsubunit of diol dehydratase reactivating factor and/or glyceroldehydratase reactivating factor, more preferably composed of a largesubunit of diol dehydratase reactivating factor and a small subunit ofdiol dehydratase reactivating factor and/or glycerol dehydratasereactivating factor, and particularly preferably composed of a largesubunit of diol dehydratase reactivating factor and a small subunit ofdiol dehydratase reactivating factor. In this case, a large subunit ofdiol dehydratase reactivating factor includes, for example, ddrA (NCBINo. AF017781) and a large subunit of glycerol dehydratase reactivatingfactor includes, for example, gdrA (NCBI No. U30903), but not limited tothese. A small subunit of diol dehydratase reactivating factor includes,for example, ddrB (NCBI No. AF017781) and a small subunit of glyceroldehydratase reactivating factor includes, for example, gdrB (NCBI No.U30903), but not limited to these. These reactivating factors may beused alone or as a mixture of two or more kinds. Further, order of alarge subunit and a small subunit is not especially limited, and may bein any order of a large subunit and a small subunit, or a small subunitand a large subunit, or, when each of these units exists in plural,these units may exist in any of block or random, but from the viewpointof a high reactivation ability, preferably a large subunit exist inupstream of a small subunit.

In the present invention, a source of the glycerol dehydratasereactivating factor is not especially limited. The factor is coded on agenome of a microorganism having glycerol dehydratase as describedabove, and composed of a large subunit and a small subunit.Specifically, glycerol dehydratase reactivating factors derived frommicroorganisms belonging to Lactobacillus genus, Klebsiella genus,Citrobacter genus, Clostridium genus and Enterobacter genus, and morespecifically, the microorganisms include, for example, Lactobacillussp., Klebsiella pneumoniae, Lactobacillus leichmannii, Citrobacterfreundii, Citrobacter intermedium, Lactobacillus reuteri, Lactobacillusbuchneri, Lactobacillus brevis, Clostridium Pasteurianum, Enterobacteragglomerans and Clostridium butyricum may be cited. Among thesemicroorganisms, microorganisms belonging to Lactobacillus genus, forexample, Lactobacillus sp., Lactobacillus leichmannii, Lactobacillusreuteri, Lactobacillus buchneri and Lactobacillus brevis may bepreferably used, and glycerol dehydratase reactivating factors derivedfrom Lactobacillus sp. and Lactobacillus reuteri are particularlypreferably used. In this connection, sources of glycerol dehydratase andglycerol dehydratase reactivating factor may be the same or differentfrom each other.

Method for isolating glycerol dehydratase reactivating factor from thesources is not especially limited, and glycerol dehydratase reactivatingfactor may be isolated from any microorganism as described abovesimilarly to a well-known separation and isolation method of enzyme suchas extraction and column chromatography.

Further, in the present invention, source of the diol dehydratasereactivating factor is not especially limited. The factor is coded on agenome of a microorganism having diol dehydratase as described above,and composed of a large subunit and a small subunit. In this connection,this glycerol dehydratase reactivating factor is preferably used becausethe factor can reactivate both of glycerol dehydratase and dioldehydratase. Specifically, diol dehydratase reactivating factors derivedfrom microorganisms belonging to Klebsiella genus, Citrobacter genus,Propionibacterium genus, Lactobacillus genus, Flavobacterium genus andAcetobacterium genus may be used. More specifically, the microorganismsinclude, for example, Klebsiella pneumoniae, Citrobacter freundii,Propionibacterium freudenreichii, Lactobacillus brevis, Lactobacillusbuchneri, Flavobacterium sp. and Acetobacterium sp. Among thesemicroorganisms, diol dehydratase reactivating factors derived frommicroorganisms belonging to Lactobacillus genus, that is, Lactobacillusbrevis and Lactobacillus buchneri, in particular, Lactobacillus breviscan be preferably used. In this connection, sources of diol dehydrataseand diol dehydratase reactivating factor may be the same or differentfrom each other, but from the viewpoint of more superior reactivationability, a large subunit of diol dehydratase reactivating factor ispreferably used. Accordingly, the combination of a large subunit of dioldehydratase reactivating factor and a small subunit of glyceroldehydratase reactivating factor and/or diol dehydratase reactivatingfactor is more preferably used.

Method for isolating diol dehydratase reactivating factor from thesources is not especially limited, and glycerol dehydratase reactivatingfactor may be isolated from any microorganism as described abovesimilarly to a well-known separation and isolation method of enzyme suchas extraction and column chromatography.

Alternatively, a recombinant microorganism containing a gene coding fordehydratase and/or dehydratase reactivating factor and a microorganismmutated so as to improve an activity of dehydratase and/or dehydratasereactivating factor may be used as a microorganism sourcehaving/expressing the dehydratase and/or dehydratase reactivatingfactor. These recombinant microorganisms can be prepared using aconventional method. For example, microorganisms expressing dehydrataseinclude, specifically, those disclosed in Journal of Bacteriology, Vol.181, No. 13, pp. 4110-411.3, 1999; The Journal of Biological Chemistry,Vol. 272, No. 51, pp. 32034-32041, 1997; Arch. Microbiol., 174:81-88(2000); The Journal of Biological Chemistry, Vol. 274, No. 6, pp.3372-3377, 1999, all of which were described in the section ofDescription of Related Art, as well as those disclosed in WO 98/21339,WO 99/58686, WO 98/21341, U.S. Pat. No. 6,025,184, WO 01/12833, WO96/35795, WO 01/04324, FEMS Microbiology Letters 164 (1998) 21-28,Applied and Environmental Microbiology, January 1998, p. 98-105, Appliedand Environmental Microbiology, December 1991, p. 3541-3546, and thelike. Also, microorganisms mutated so as to improve an activity ofdehydratase and/or dehydratase reactivating factor can be prepared usinga conventional method, and include, specifically, for example, thosedisclosed in WO 00/70057 and the like.

In the present invention, 3-hydroxypropionaldehyde can be produced bydehydration of glycerin using microbial cell and/or treated microbialcell containing diol/glycerol dehydratase and, if necessary,diol/glycerol dehydratase reactivating factor. In this case, from theviewpoints that a very high conversion ratio can be attained asdescribed later in detail, and 3-hydroxypropionaldehyde obtainedcontains less amount of by-products, preferably “microbial cell” and“treated microbial cell” do not use glycerin as an energy source of themicroorganism. In the present invention, treated microbial cell is morepreferably used. This is because use of treated microbial cell allowsconversion of glycerin to 3-HPA by a method other than fermentation andthus attainment of high conversion ratio and low content of by-product.

In this connection, in the present invention, “microbial cell” is notespecially limited so long as the microbial cell has diol dehydrataseand/or glycerol dehydratase, and, if necessary, diol dehydratasereactivating factor and/or glycerol dehydratase reactivating factor, andan ability to convert glycerin to 3-HPA. It does not matter whether amicrobial cell has a proliferating ability or not when the microbialcell is returned to a suitable culture medium after completion of thereaction of the present invention. Preferably, microbial cell is one inwhich 3-HPA is not further used to another reaction, and which allowsconversion of glycerin to 3-HPA in a reaction system containing glycerinby a method other than fermentation, that is, does not assimilateglycerin nor proliferate using glycerin as an energy source. Forexample, these microbial cells include preferably a microbial cellobtained by culturing a microbial cell having an ability to convertglycerin to 3-HPA under aerobic conditions; a microbial cell obtained byculturing the microbial cell under conditions so as not to produce NADHand/or NADPH; and a recombinant microbial cell obtained by introducingdiol/glycerol dehydratase or diol/glycerol dehydratase reactivatingfactor to a suitable host, and more preferably a microbial cell whichhas been genetically manipulated so as to have a plurality of copies ofdiol/glycerol dehydratase to highly express these enzymes and the like.Further, “fermentation” means an activity of microorganism performing,in a reaction system in which 3-hydroxypropionaldehyde is obtained fromglycerin, at a same time, conversion of glycerin and/or proliferationusing glycerin as an energy source and/or oxidation of glycerin and thelike. Hence, in the present invention, “method other than fermentation”means a reaction method in which 3-hydroxypropionaldehyde is formed bydehydration of glycerin in a reaction system containing glycerin of thepresent invention, but at the same time not being accompanied byconversion of glycerin, proliferation using glycerin as an energy andoxidation of glycerin.

Herein, “treated microbial cell” means a microbial cell which has beentreated so as to be easily used for the reaction of the presentinvention. Specifically, examples of the treated microbial cell include,for example, immobilized substance (for example, immobilized enzyme)such as immobilized dehydratase or immobilized dehydratase reactivatingfactor as described above; toluene-treated microbial cell which isobtained by treating microbial cell containing dehydratase ordehydratase reactivating factor as described above with an organicsolvent such as toluene; and immobilized microbial cell of a microbialcell having dehydratase or dehydratase reactivating factor as describedabove. Among these, toluene-treated microbial cell and immobilizedmicrobial cell are preferably used, and toluene-treated microbial cellis particularly preferable. Immobilizing method in producing aimmobilized factor is not especially limited, and well-known methodssuch as a method by which a factor is immobilized on an insolublesubstrate via covalent bond, ionic bond, adsorption or the like; amethod by which a factor is cross-linked each other; and a method bywhich a factor is included in a network structure of a polymer, may beused.

Also, immobilizing method for producing an immobilized microbial cell isnot especially limited, and conventional methods such as a method bywhich microorganism having dehydratase or dehydratase reactivatingfactor as described above is supported on an insoluble substrate; amethod by which the microorganism is immobilized by being absorbed orincluded in a gel matrix; and a method by which the microorganism isentrapped in an internal space, may be used.

Further, production method for the toluene-treated microbial cell isalso not especially limited. Conventional methods such as a method whichcomprises adding toluene to a microorganism having dehydratase ordehydratase reactivating factor as described above and stirring themixture, thereby forming holes having such a size that an enzyme and afactor can not go out from the microbial cell may be used. In themethod, though toluene was used to produce a toluene-treated microbialcell, another organic solvent such as acetone, hexane and ethyl acetatemay be used instead of toluene. Among these, toluene is preferably used.In this case, amount of the organic solvent to be added is preferably inthe range of 0.1 to 10% by mass, and more preferably 0.2 to 5% by mass.Further, the toluene-treated microbial cell is not especially limited,and can be prepared by conventional methods. For example, the followingmethod can be used: A microbial cells cultured under suitable conditionsare suspended in a buffer, and toluene is added to this suspension so asto give an adequate concentration, preferably a final concentration of1% (v/v), then the suspension is vigorously stirred for a prescribedtime, preferably about 5 minutes using a vortex mixer or the like toperform toluene treatment, subsequently the microbial cell after thetoluene treatment is washed with a buffer. In the method, buffer is notespecially limited, and well-known buffers can be used. 50 mM potassiumphosphate buffer (25 ml of 1 M K₂HPO₄ and 100 ml of 1 M K₂HPO₄ aremixed, pH is adjusted at 8, then water is added to 25 ml of the solutionto make 500 ml as a whole) is particularly preferably used.

In the present invention, immobilized microbial cell and immobilizedfactor may be used in any form, and specifically, used, for example, ina form of membrane-like, particulate, ribbon-like and folded layer-like.In view of easiness in handling, ribbon-like and particle-like arepreferably used.

By using microbial cell and/or treated microbial cell, stabilization ofmicrobial cell and/or treated microbial cell containing dehydratase anddehydratase reactivating factor can be performed. In particular, whentreated microbial cell is used, in addition to an advantage that thesemicrobial cell can be continuously and repeatedly used, anotheradvantage exists that purity and yield of the desired3-hydroxypropionaldehyde can be improved because glycerin can beconveniently converted to 3-hydroxypropionaldehyde without using aculture medium.

In the present invention, method for converting glycerin to 3-HPA is notespecially limited, and a method similar to conventionally known methodscan be used using a factor, an immobilized microbial cell and animmobilized factor. Differing from the case of fermentation, since theconversion of glycerin to 3-HPA according to the present invention iscarried out using microbial cell and/or treated microbial cell (forexample, toluene-treated microbial cell and immobilized microbial cell),3-hydroxypropionaldehyde to be obtained scarcely contains by-product,and further, since almost all of glycerin is used for the reaction, ahigh conversion ratio can be attained. Further, in comparing withconventional chemical method, since raw material is mainly glycerin, andboth the conversion ratio and selectivity to 3-hydroxypropionaldehydeare very high and purity is also high, a purification process to removeby-product can be performed conveniently, and the method is veryadvantageous from the economical viewpoint.

A preferable embodiment of the conversion of glycerin to 3-HPA accordingto the present invention will be described below. For example, when aparticulate immobilized microbial cell and/or immobilized factor isused, it is added into a mixed solution containing a suitable buffer(for example, potassium phosphate buffer), an appropriate amount ofcoenzyme B12 as described above and glycerin, so that a enzyme asdescribed above is suitably present, and then the resultant mixture isstirred at 10 to 90° C., preferably at 15 to 85° C. for 1 to 360minutes, preferably for 5 to 120 minutes to form 3-HPA. The 3-HPA thusformed is in a mixed state with immobilized microbial cell on theparticle, and can be easily separated from the immobilized microbialcell by a conventional method such as filtration, ultrafiltration andsettling. Alternatively, 3-HPA may be formed by passing a mixed solutioncontaining a suitable buffer (for example, potassium phosphate buffer),an appropriate amount of coenzyme B12 as described above, glycerin andthe like through a column packed with a particulate immobilizedmicrobial cell and/or immobilized factor at 10 to 90° C., preferably at15 to 85° C., and at a flow rate of 0.1 to 50 LHSV, preferably 0.2 to 40LHSV. In this case, glycerin concentration is not especially limited, solong as the factor can sufficiently act thereon, and it is varied with acatalytic amount of diol dehydratase and/or glycerol dehydratase, andalso may be such a concentration at which X/Y² is not fallen within arange of 10 to 8,000. Preferably, it is in the range of 0.1 to 50%(w/v), more preferably 0.2 to 40% (w/v). Further, in the method, liquidused for dissolving glycerin is not especially limited so long asglycerin can be dissolved. Water, and various buffers such asphysiological saline, potassium phosphate buffer, potassium citratebuffer, phosphate buffer, Good's buffer and Tris buffer may be used, forexample. Among these, water, potassium phosphate buffer and phosphatebuffer are preferably used. In the present invention, presence ofpotassium ion in a reaction system is preferable in view of an activityof enzyme. For the presence of potassium ion in the reaction system,buffers containing potassium salts such as potassium phosphate buffer,potassium citrate buffer and other potassium salt aqueous solution isparticularly preferably used as a reaction medium. Concentration ofpotassium ion is not especially limited, but preferably in the range of.5 mM to 1 M, and more preferably 10 to 500 mM.

According to the method of the present invention, the 3-HPA thusobtained is, after microbial cell and/or treated microbial cell beingremoved, hydrogenated to form desired 1,3-propanediol. Thus, a secondaspect of the present invention relates to a method for producing1,3-propanediol which comprises a step of removing the microbial celland/or treated microbial cell from the 3-hydroxypropionaldehyde producedby the method of the present invention, subsequently hydrogenating said3-hydroxypropionaldehyde to produce 1,3-propanediol. According to themethod of the present invention, since the conversion from glycerin to3-HPA occurs preferentially in a high conversion ratio,3-propionaldehyde of the product can be produced in a high puritywithout containing not only glycerin as a substrate but also anyby-product. Accordingly, by using 3-HPA of the present invention, a highpurity of 3-HPA can be obtained by a simple procedures to separate orremove microbial cell/treated microbial cell. Further, by hydrogenatingthis 3-HPA, 1,3-propanediol can also be produced in a high purity.

In the present invention, separation/removal of microbial cell is notespecially limited, and performed by a conventional method.Specifically, by using a known method such as filtration,ultrafiltration and settling, immobilized microbial cell can be easilyseparated from 3-HPA.

In the present invention, hydrogenation may be performed by any offermentation or chemical synthesis method, but chemical synthesis methodis preferable. This is because there is an advantage that by producing1,3-propanediol from 3-HPA using chemical synthesis method, purity andyield of desired 1,3-propanediol can be improved. In this case, in thechemical synthesis method, conventional hydrogenation method may beused, and the reaction may be carried out either in a gas phase or aliquid phase. 3-HPA is hydrogenated preferably in a liquid phase, andmore preferably in an aqueous solution, to form desired 1,3-propanediol.The chemical synthesis method to produce 1,3-propanediol from 3-HPA isnot especially limited, and conventional methods can be used. Forexample, the methods include, for example, a method which comprisesadding palladium carbon to 3-HPA, replacing a gas phase part withhydrogen, and hydrogenating the 3-HPA with hydrogen while stirring; amethod which comprises hydrogenating 3-HPA in the presence of aheterogeneous catalyst having ruthenium supported on an oxide carrier ata temperature of 30 to 180° C. and under a hydrogen pressure of 5 to 300bar, in an aqueous solution of pH value 2.5 to 7.0 (for example,JP-A-2002-516614); a method which comprises hydrogenating 5 to 100% byweight of 3-HPA in an aqueous solution, on a catalyst having platinumsupported on titanium oxide as a carrier, at a temperature of 30 to 180°C., at pH value of 2.5 to 6.5, and under a hydrogen pressure of 5 to 300bar (for example, JP-A-5-213800); and a method which comprisescatalytically hydrogenating 3-HPA in an aqueous solution in the presenceof a catalyst such as a catalyst having Pt supported on TiO₂ and aNi/Al₂O₃/SiO₂-catalyst, under a hydrogen pressure of 5 to 300 bar, at pHvalue of 2.5 to 6.5, and at a temperature of 30 to 180° C. (for example,JP-A-6-40973).

According to the method of the present invention, 1,3-propanediol andwater are mainly formed, and other by-products and secondary productsare scarcely present. Hence, purification consists mainly of removal ofwater only, not complicated, and when fiber is produced using this1,3-propanediol as a raw material thereafter, there is no risk resultingdiscoloration and defective polymerization in a product such as fiber.In this case, purification method of 1,3-propanediol to be used is notespecially limited, and conventional methods can be used. For example, amethod such as by distillation and reverse osmosis membrane can be used.

According to the present invention, by oxidizing3-hydroxypropionaldehyde obtained similarly as above, 3-hydroxypropionicacid is produced. Thus, a third aspect of the present invention relatesto a method which comprises a step of oxidizing the3-hydroxypropionaldehyde produced by the method of the present inventionto produce 3-hydroxypropionic acid.

In the present invention, in view of purity of 3-hydroxypropionic acid,microbial cell/treated microbial cell has been preferably separated orremoved in advance prior to oxidation of 3-hydroxypropionaldehyde. Inthis case, separation/removal of microbial cell/treated microbial cellis not especially limited, but may be performed by conventional methods.Specifically, by using known methods such as filtration, ultrafiltrationand settling, immobilized microbial cell can be removed from 3-HPA.

In the present invention, although production of 3-hydroxypropionic acidfrom 3-HPA may be performed by any of fermentation or chemical synthesismethod, in view of purity and yield of 3-hydroxypropionic acid to beproduced, chemical synthesis method is preferably used. In the oxidationaccording to the present invention, conventional oxidation methods canbe used, and the reaction may be carried out either in a gas phase or aliquid phase. 3-HPA is oxidized preferably in a liquid phase, and morepreferably in an aqueous solution. Method to be used in this case is notespecially limited, conventional methods such as a method using platinumcarbon, palladium carbon, or the like can be used. Specifically, themethod includes a method which comprises adding palladium carbon to3-HPA, and replacing a gas phase part with hydrogen, and oxidizing the3-HPA with oxygen while stirring; and a method which comprises addingplatinum carbon and sodium bicarbonate as catalysts to 3-HPA reactionliquid and contacting the 3-HPA with oxygen to oxidize the 3-HPA.

According to the method of the present invention, 3-hydroxypropionicacid can be mainly formed. For this reason, purification is easy. Inthis case, purification is not necessarily required. Purification methodfor 3-hydroxypropionic acid before use is not especially limited, andconventional methods, for example, a method such as distillation andreverse osmosis membrane can be used.

Further, according to the present invention, through a reaction of3-hydroxypropionaldehyde obtained similarly as described above underacidic conditions, acrolein can be produced. Thus, a fourth aspect ofthe present invention relates to a method for producing acrolein whichcomprises a step of reacting the 3-hydroxypropionaldehyde produced bythe method of the present invention under acidic conditions, to produceacrolein. Further, according to the present invention, through anoxidation of acrolein obtained by the fourth aspect of the presentinvention, acrylic acid is produced. Thus, a fifth aspect of the presentinvention relates to a method for producing acrylic acid which comprisesa step of oxidizing the acrolein produced by the method of the presentinvention to produce acrylic acid.

In the fourth and fifth aspects of the present invention, in view ofpurities of acrolein and acrylic acid as final products, microbialcell/treated microbial cell has been preferably separated or removed inadvance prior to oxidation of 3-hydroxypropionaldehyde. In this case,separation/removal of microbial cell/treated microbial cell is notespecially limited, but may be performed by conventional methods.Specifically, by using known methods such as filtration, ultrafiltrationand settling, immobilized microbial cell can be removed from 3-HPA.

In the present invention, production of acrolein from 3-HPA can beeasily attained through a reaction of 3-HPA under acidic conditions. Forexample, acrolein can be efficiently produced by adding/mixing an acidsuch as hydrochloric acid, sulfuric acid and nitric acid, preferablyhydrochloric acid, to a solution containing 3-HPA so as to give a pHvalue of 1 to 5, preferably 1.5 to 4.5, then standing at 5 to 90° C.,preferably at 7 to 85° C. for 1 to 360 minutes, preferably 2 to 120minutes. Since no components other than acid are used in this process,by-product is scarcely formed. Since the reaction has a high conversionratio and a high selectivity, acrolein can be produced in a high purityand in a high yield.

In the present invention, acrolein thus formed is oxidized to produceacrylic acid. Production method from acrolein to acrylic acid is notespecially limited, and may be any of fermentation or chemical synthesismethod. However, in view of purity and yield of acrylic acid to beproduced, chemical synthesis method is preferably used. In the oxidationaccording to the present invention, conventional oxidation methods canbe used, and the reaction may be carried out either in a gas phase or aliquid phase. Further, oxidation method for acrolein in liquid phase isnot also especially limited, and conventional methods can be used. Forexample, a method for producing acrylic acid by oxidizing acrolein inthe presence of a palladium carbon catalyst while oxygen is added toreaction liquid containing acrolein, can be used. Further, oxidationmethod for acrolein in gas phase is not especially limited, andconventional methods can be used. For example, known methods such asdisclosed in JP-A-64-63543 and JP-A-63-146841 can be used. Specifically,catalyst to be used for oxidizing acrolein to acrylic acid is notespecially limited, and conventional catalysts can be used alone or incombination thereof. For example, catalysts containing molybdenum andvanadium, and preferably a catalyst represented by the general formula:Mo_(a)—V_(b)—W_(c)—Cu_(d)-A_(e)-B_(f)—C_(g)—O_(x) (wherein Mo representsmolybdenum, V represents vanadium, W represents tungsten, Cu representscopper, A represents at least one element selected among antimony,bismuth, tin, niobium, cobalt, iron, nickel and chromium, B representsat least one element selected among alkali metals and alkaline earthmetals, C represents at least one element selected from silicon,aluminum, zirconium and titanium, O represents oxygen, each of a, b, c,d, e, f, g and x represents atomic ratio of Mo, V, W, Cu, A, B, C and O,respectively, provided that when a=12, then b=2 to 14, c=0 to 12, d=0.1to 5, e=0 to 5, f=0 to 5, g=0 to 20, and x is a value depending onoxidation state of each element) may be cited. Further, preparationmethod and mixing and molding method of catalyst to be used in this caseare also not especially limited, and methods and raw materials whichhave been generally used in the art can be employed. Further, form ofcatalyst is also not especially limited, and for example, spherical,cylindrical and column-shaped forms can be used. As for forming method,support forming, extrusion forming, tablet compression, and the like canbe used, and still further, a catalyst having these catalyst materialssupported on a refractory carrier is also useful.

Further, reaction conditions used in the conversion from acrolein toacrylic acid is also not especially limited. For example, the reactionis performed by mixing acrolein with oxygen needed for conversion toacrylic acid and steam, and feeding this acrolein-containing gas under areaction pressure in a range of atmospheric to 0.5 MPa, at a spacevelocity in a range of 300 to 5,000 hr⁻¹ (STP), with a reactiontemperature being controlled at 200 to 400° C., and preferably 220 to380° C.

The acrylic acid thus obtained is recovered by a common method. Forexample, the acrylic acid forming gas is, after cooled with a heatexchanger, made in countercurrent contact with a catching solventcontaining a polymerization inhibitor to obtain acrylic acid aqueoussolution, from which acrylic acid is isolated by a method such asextraction, distillation and azeotropic distillation.

Further, in the present invention, acrolein obtained in the fourthaspect is then converted to acrylic esters via oxidative esterificationin the presence of a catalyst. Thus, a sixth aspect of the presentinvention relates to a method for producing an acrylic ester whichcomprises a step of subjecting the acrolein produced by the method ofthe present invention, to produce an acrylic ester. In this case,production method of an acrylic ester from acrolein is not especiallylimited, and any of fermentation or chemical synthesis method can beused. However, in view of purity and yield of an acrylic ester to beproduced, chemical synthesis method is preferably used. In the oxidationaccording to the present invention, conventional oxidation methods canbe used, and the reaction may be carried out either in a gas phase or aliquid phase. The reaction is performed particularly preferably in aliquid phase in the presence of a catalyst. In this case, as a catalyst,the similar catalyst as used in the production of acrylic acid can beused. Further, conditions of reaction from acrolein to acrylic ester isalso not especially limited, and for example, the similar conditions asused in the production of acrylic acid can be used. The thus obtainedacrylic ester is recovered by a common method.

EXAMPLES

The present invention will be explained more specifically with referringto working examples below.

Example 1

A strain JM109/vector 1 (DD)/vector 2 (DDR), which was obtained bytransforming E. coli JM 109 as a host with vector 1 (FIG. 1A, SEQ IDNO: 1) obtained by inserting a gene coding for diol dehydratase ofKlebsiella pneumoniae (ATCC 25955) into a plasmid having a replicationorigin (ori) derived from pBR322 and vector 2 (FIG. 1B, SEQ ID NO: 2)obtained by inserting a gene coding for diol dehydratase reactivatingfactor of Klebsiella pneumoniae (ATCC 25955) into a plasmid having areplication origin (ori) derived from p15A, was inoculated into a LBmedium containing 50 μg/ml of ampicillin and 100 μg/ml ofchloramphenicol, and cultured at 37° C. for 15 hours. The culturedliquid was inoculated into 200 ml of a LB medium containing 50 μg/ml ofampicillin and 100 μg/ml of chloramphenicol, and cultured with shakingat 37° C. When an absorbance at 660 nm reached 0.8 (OD=0.8) afterinitiation of the culture, IPTG was added so as to give a concentrationof 1 mM, and cultured for further 5 hours, then the culture was stopped.Microbial cells were collected by centrifugation. Collected cells werewashed twice with 50 mM of potassium phosphate buffer (pH 8), andsuspended in 50 mM potassium phosphate buffer (pH 8) so as to giveOD660=0.2. This microbial cell suspending fluid was used as a crudeenzyme fluid. SEQ ID NO: 1 and SEQ ID NO: 2 show base sequences ofvector 1 and vector 2 used in the present Example.

To 4 ml of the crude enzyme fluid, 3 ml of 50 mM potassium phosphatebuffer (pH 8), 1 ml of 2 M glycerin, 1 ml of 0.5 M KCl and 1 ml of 150μM coenzyme B12 were added, and the mixture was reacted at 37° C. for 60minutes. An aliquot of the reaction liquid was taken and this aliquot istested for quantitative determination of 3-hydroxypropionaldehydetherein. An equivalent amount of 0.1 M potassium citrate buffer (pH 3.0)was added to the reaction liquid to stop the reaction. An equivalentamount of water was added thereto, and 0.5 times by volume of an aqueoussolution of 0.1% 3-methyl-2-benzothiazolinon hydrazone hydrochloridemonohydrate was added, absorbance at 305 nm was measured to determine aconcentration of 3-hydroxypropionaldehyde. The result indicated that 0.1M 3-hydroxypropionaldehyde was formed in the reaction mixture. Theresidual reaction mixture was filtered to remove microbial cells. Afterthe filtrate was placed into a test tube with a sealed cap, 0.015 g of5% palladium carbon was added thereto, and a gas phase part was replacedwith hydrogen gas. A balloon was filled with 500 ml of hydrogen gasunder normal pressure, connected to the gas phase part and sealed, andthe reaction was carried out with stirring at 60° C. for 5 hours in ahot-water bath. Analysis of the reaction mixture indicated that 0.098 Mof 1,3-propandiol was formed. In this case, catalyst amount of microbialcells [X (U/g glycerin)], concentration of glycerin [Y (g/100 ml)], andX/Y² are summarized in Table 1 below. In Table 1, the values of [X (U/gglycerin)], [Y (g/100 ml)], and X/Y² disclosed in the documents listedas related references in the section of Description of Related Art arealso summarized together in Table 1 below.

Example 2

A strain JM109/vector 1′ (GD)/vector 2′ (GDR), which was obtained bytransforming E. coli JM 109 as a host with vector 1′ (FIG. 2A, SEQ IDNO: 3) obtained by inserting a gene coding for glycerol dehydratase ofKlebsiella pneumoniae (ATCC 25955) into a plasmid having replicationorigin (ori) derived from pBR322 and vector 2′ (FIG. 2B, SEQ ID NO: 4)obtained by inserting a gene coding for glycerol dehydratasereactivating factor of Klebsiella pneumoniae (ATCC 25955) into a plasmidhaving replication origin (ori) derived from p15A, was inoculated into aLB medium containing 50 μg/ml of ampicillin and 100 μg/ml ofchloramphenicol, and cultured at 37° C. for 15 hours. The culturedliquid was inoculated into 200 ml of a LB medium containing 50 μg/ml ofampicillin and 100 μg/ml of chloramphenicol, and cultured with shakingat 37° C. When an absorbance at 660 nm reached 0.8 (OD=0.8) afterinitiation of the culture, IPTG was added so as to give a concentrationof 1 mM, and cultured for further 5 hours, then the culture was stopped.Microbial cells were collected by centrifugation. Collected cells werewashed twice with 50 mM of potassium phosphate buffer (pH 8), andsuspended in 50 mM potassium phosphate buffer (pH 8) so as to giveOD660=0.2. This microbial cell suspending fluid was used as a crudeenzyme fluid. Toluene was added to the suspension so as to give a finalconcentration of 1%. After the mixture was stirred by using a vortexmixer for 5 minutes, microbial cells were collected by centrifugation.Microbial cells were suspended into 50 mM potassium phosphate buffer (pH8) so as to give OD660 of 0.2. This suspension was designated as atoluene-treated microbial cell fluid. In this connection, SEQ ID NO: 3and SEQ ID NO: 4 show base sequences of vector 1′ and vector 2′ used inthe present Example.

To the toluene-treated microbial cell fluid, 3 ml of 50 mM potassiumphosphate buffer (pH 8), 1 ml of 2M glycerin, 1 ml of 0.5 M KCl and 1 mlof 150 μM coenzyme B12 were added, and the mixture was reacted at 37° C.for 20 minutes. Analiquot of the reaction liquid was taken and thisaliquot is tested for quantitative determination of3-hydroxypropionaldehyde therein. An equivalent amount of 0.1 Mpotassium citrate buffer (pH 3.0) was added to the reaction liquid tostop the reaction. An equivalent amount of water was added thereto, and0.5 times by volume of an aqueous solution of 0.1%3-methyl-2-benzothiazolinon hydrazone hydrochloride monohydrate wasadded, absorbance at 305 nm was measured to determine a concentration of3-hydroxypropionaldehyde. As a result, it was confirmed that 0.196 M of3-hydroxypropionaldehyde was formed in the reaction mixture. Aconversion ratio of glycerin to 3-hydroxypropionaldehyde was 98%. Theresidual reaction mixture was filtered to remove toluene-treatedmicrobial cells. After the filtrate was placed into a test tube with asealed cap, 0.015 g of 5% palladium carbon was added thereto, and a gasphase part was replaced with hydrogen gas. A balloon was filled with 500ml of hydrogen gas under normal pressure, connected to the gas phasepart and sealed, and the reaction was carried out with stirring at 60°C. for 5 hours in a hot-water bath. Analysis of the reaction mixtureindicated that 0.196 M of 1,3-propandiol was formed. In this case,catalyst amount of microbial cells [X (U/g glycerin)], concentration ofglycerin [Y (g/100 ml)], and X/Y² are shown in Table 1 below.

Example 3

In the same manner as in Example 2, 0.196 M of 3-hydroxypropionaldehyde(conversion ratio of glycerin: 98%) was produced. The reaction mixturecontaining the same was adjusted to pH 2 with 35% hydrochloric acid, andallowed to stand at room temperature for 1 hour. The acrolein formed inthe reaction mixture was quantitatively determined, to find that 0.130 Mof acrolein was formed.

Example 4

In the same manner as in Example 1, 0.188 M of 3-hydroxypropionaldehyde(conversion ratio of glycerin: 94%) was produced. After the reactionmixture was placed into a test tube with a sealed cap, 0.015 g of 5%palladium carbon was added thereto, and a gas phase part was replacedwith oxygen gas. A balloon was filled with 1 litter of oxygen gas undernormal pressure, connected to the gas phase part and sealed, and thereaction was carried out with stirring at 60° C. for 5 hours. Analysisof the reaction mixture indicated that 0.150 M of 3-hydroxypropionicacid was formed.

Example 5

In the same manner as in Example 2, 0.196 M of 3-hydroxypropionaldehyde(conversion ratio of glycerin: 98%) was produced. 10 ml of the reactionmixture containing the same was adjusted to pH 2 with 35% hydrochloricacid, and allowed to stand at room temperature for 1 hour. The acroleinformed in the reaction mixture was quantitatively determined, to findthat 0.150 M of acrolein was formed.

Subsequently, methanol was added to the resultant acrolein. The reactionwas performed with stirring thoroughly under an oxygen atmosphere using0.015 g of 5% palladium carbon as an oxidation catalyst, to form 0.188 Mof methyl acrylate.

Example 6

In the same manner as in Example 2, 0.196 M of 3-hydroxypropionaldehyde(conversion ratio of glycerin: 98%) was produced. The reaction mixturecontaining the same was adjusted to pH 2 with 35% hydrochloric acid, andallowed to stand at room temperature for 1 hour. The acrolein formed inthe reaction mixture was quantitatively determined, to find that 0.148 Mof acrolein (conversion ratio to 3-hydroxypropionaldehyde: 96.9%) wasformed.

Subsequently, after the reaction mixture containing the resultantacrolein was placed into a test tube with a sealed cap, 0.015 g of 5%palladium carbon was added thereto, and a gas phase part was replacedwith oxygen gas. A balloon was filled with 1 litter of oxygen gas undernormal pressure, connected to the gas phase part and sealed, and thereaction was carried out with stirring at 60° C. for 5 hours. Analysisof the reaction mixture indicated that 0.120 M of acrylic acid wasformed.

Example 7

Klebsiella pneumoniae (ATCC 25955) was anaerobically cultured in amedium containing glycerin as a carbon source to grow until reaching alogarithmic growth phase. The microbial cell culture at this stage wastreated with 1% toluene in the same manner as described in Example 2,and the treated microbial cells were collected. The collected microbialcells of 20 g (wet weight, enzymatic activity 4000 U) of Klebsiellapneumoniae (ATCC 25955) were added to l litter of 50 mM potassiumphosphate buffer (pH 8) containing 135 μM coenzyme B12 and 0.2 Mglycerin, and the resultant mixture was reacted at 37° C. for 120minutes. After the reaction mixture was filtered to remove off thetoluene-treated microbial cells, an aliquot thereof was taken out and anamount of 3-hydroxypropionaldehyde therefor was quantitativelydetermined to find that 0.196 M of 3-hydroxypropionaldehyde was formed.The resultant 3-hydroxypropionaldehyde was placed into a 1 litter ofseparable flask, 1.5 g of 5% palladium carbonawas added thereto, and agas phase part was replaced with oxygen. A small amount of oxygen waspassed through the gas phase part in order to prevent the penetration ofoutside gas. The mixture was reacted at 60° C. for 5 hours withstirring. The reaction mixture was analyzed to detect 0.178 M of3-hydroxypropionic acid. Catalyst amount of the toluene-treatedmicrobial cells [X (U/g glycerin)], concentration of glycerin [Y (g/100ml)], and X/Y² are summarized in Table 1 below.

Example 8

Klebsiella pneumoniae (ATCC 25955) was anaerobically cultured in amedium containing glycerin as a carbon source to grow until reaching alogarithmic growth phase. The microbial cell culture at this stage wastreated with 1% toluene in the same manner as described in Example 2,and the treated microbial cells were collected. The collected microbialcells of 20 g (wet weight, enzymatic activity 4000 U) of Klebsiellapneumoniae (ATCC 25955) were added to 1 litter of 50 mM potassiumphosphate buffer (pH 8) containing 135 μM coenzyme B12 and 0.2 Mglycerin, and the resultant mixture was reacted at 37° C. for 120minutes. After the reaction mixture was filtered to remove off thetoluene-treated microbial cells, an aliquot thereof was taken out and anamount of 3-hydroxypropionaldehyde therefor was quantitativelydetermined to find that 0.197 M of 3-hydroxypropionaldehyde was formed.The resultant reaction mixture containing 3-hydroxypropionaldehyde wasadjusted to pH 2 with 35% hydrochloric acid, and allowed to stand atroom temperature for 1 hour. An amount of acrolein formed in thereaction mixture was quantitatively determined, to find that 0.130 M ofacrolein was formed. Catalyst amount of the toluene-treated microbialcells (X (U/g glycerin)), concentration of glycerin (Y (g/100 ml)), andX/Y² are summarized in Table 1 below.

[Table 1]

TABLE 1 Catalytic Substrate amount X conc. Y Conversion Source (U/g Gly)(%) X/Y² (%) J. Bac. Vol. 181, No. 13, 8 0.92 8.989 1.4 '99, p.4110-4113 The J. of Biolog. Chem., 1 1.84 0.273 0.17 Vol. 272, No. 51,'97 Arch. Microbiol., 174, 8 0.92 8.989 1.4 81-88 (2000) The J. ofBiolog. Chem., 1 11.04 0.010 0.27 Vol. 274, No. 6, '99 The J. of Biolog.Chem., 7 13.8 0.034 0.75 Vol. 274, No. 6, '99 Example 1 91 1.84 27 50Example 2 533 1.84 157 98 Example 3 533 1.84 157 98 Example 7 217 1.8464 98 Example 8 217 1.84 64 98.5

Example 9

To 10 ml of 50 mM potassium phosphate buffer (pH 8) containing 4.6% (0.5M) of glycerin as a substrate and 135 μM of coenzyme B12, an enzymaticactivity 200 U/g wet weight of toluene-treated (Klebsiella pneumoniae)microbial cells were added so as to give a catalyst amount as shown inTable 2 below. The mixture was reacted with stirring at 37° C. for 6hours in dark. The toluene-treated microbial cells were removed off bycentrifugation of the reaction mixture after performing the reaction fora prescribed time. Then, the conversion ratio from glycerin to3-hydroxypropionaldehyde was determined using the supernatant. Catalystamount of the toluene-treated microbial cells [X (U/g glycerin)],concentration of glycerin [Y (g/100 ml)], and X/Y² are summarized inTable 2 below.

[Table 2]

TABLE 2 Amount of Catalytic Substrate microbial amount X conc. YConversion cells (g) (U/g glycerin) (%) X/Y² (%) 0.1 920 4.6 43 52 0.21840 4.6 87 89 0.3 1304 4.6 95 96 0.5 2174 4.6 103 98 1 4348 4.6 205 982 8696 4.6 411 98 5 21739 4.6 1027 97 10 43478 4.6 2055 98 15 65217 4.63082 95 20 86957 4.6 4109 95 25 108696 4.6 5137 90 30 130435 4.6 6164 8535 152174 4.6 7192 70 40 173913 4.6 8219 65 45 195652 4.6 9146 51 50217391 4.6 10274 30

As shown in the results of the Table 2, when X/Y² exceeds 80, a highconversion ratio as of about not less than 90% can be achieved. When thevalue is not more than 8219, it is shown that the conversion ratio isalso below 70%. Further, when X/Y² is within a range of 87 to 6164, aconversion ratio of not less than 80% can be achieved, especially whenX/Y² is in a range of 95 to 5137, it is shown that an extremely highconversion ratio not less than 80% can be achieved.

Example 10

A toluene-treated microbial cell culture of a strain JM 109/vector 1′(GD) was prepared by the same way as described in Example 2. Thetoluene-treated microbial cells (enzymatic activity 9, 900 U/g wetweight) was added into 50 mM potassium phosphate buffer (pH 8)containing 9.2% (1 M) of glycerin as a substrate and 135 μM of coenzymeB12 so as to give a catalytic amount as shown in Table 3 below, and thetotal volume was adjusted to 10 ml. The mixture was reacted at 37° C.for 2 hours in dark. The toluene-treated microbial cells were removedoff by centrifugation of the reaction mixture after performing thereaction for a prescribed time, and a conversion ratio from glycerin to3-hydroxypropionaldehyde was determined using the supernatant. Catalystamount of the toluene-treated microbial cells [X (U/g glycerin)],concentration of glycerin [Y (g/100 ml)], and X/Y² are summarized inTable 3 below.

[Table 3]

TABLE 3 Amount of Catalytic Substrate microbial amount X conc. (Y)Conversion cells (g) (U/g glycerin) (%) X/Y² (%) 0.051 543 9.2 6 43.80.101 1087 9.2 13 72.1 0.202 2174 9.2 26 79.3 0.303 3261 9.2 39 79.40.404 4348 9.2 51 83.3 0.752 8096 9.2 77 78.3

As shown in the result of the Table 3, when X/Y² exceeds 10, a highconversion ratio as of not less than 70% can be achieved. Especiallywhen X/Y² exceeds 50, it is shown that an extremely high Conversionratio as of not less than 80% can be achieved. Further, it is noted inthis Example that even if a considerably high concentration of substrateas high as 9.2% is used, an conversion ratio of not less than 70% can beachieved by applying an action of the toluene-treated microbial cells toglycerin under conditions that X/Y² becomes not less than 10. Inaddition, as shown in this Example, according to the method of thepresent invention, since a high conversion ratio can be achieved even ina high concentration of substrate, it is suggested that the method ofthe present invention is very advantageous from the viewpoint of anindustrial level.

INDUSTRIAL APPLICABILITY

According to the method for producing 3-hydroxypropionaldehyde of thepresent invention, by acting diol/glycerol dehydratase and/ordiol/glycerol dehydratase reactivating factor on 3-HPA while an amountof enzyme being controlled within a specified range, the reaction fromglycerin to 3-HPA occurs selectively without inducing any side reactionother than the reaction from glycerin to 3-HPA, such a high conversionratio as not less than 80%, in some cases, not less than 90% can beattained, and 3-hydroxypropionaldehyde can be produced in a high yield.Further, in the method of the present invention, since the reaction fromglycerin to 3-HPA can be performed without using a fermentation method,3-hydroxypropionaldehyde can be produced with a high purity scarcelycontaining by-product. Accordingly, 3-HPA obtained by the method of thepresent invention can be used as an intermediate in producing1,3-propanediol by hydrogenation, through a very simple purificationprocess for separation of microbial cell/treated microbial cell such asfiltration, ultrafiltration and settling, and the like. In addition tothe advantage, since only 1,3-propanediol and water mainly remain aftercompletion of the reaction, and no organic solvents are needed forpurification, the method does not require recovery of organic solventand post-treatment, which is preferable from the viewpoint ofenvironment.

Further, the present invention relates to a method for obtainingacrolein by reacting the 3-hydroxypropionaldehyde scarcely containingby-product as described above under acidic conditions, and further forproducing acrylic acid by oxidizing the acrolein; and a method forproducing an acrylic ester by reacting 3-hydroxypropionaldehyde scarcelycontaining by-product as described above under acidic conditions toobtain acrolein, and further subjecting the acrolein to oxidativeesterification. According to the method, since 3-hydroxypropionaldehydeas a raw material scarcely contains by-product, acrolein produced usingthis, and further acrylic acid and an acrylic ester produced from thishave also high purities.

According to the present invention, since 3-HPA can be produced in ahigh conversion ratio and a high purity ratio, and scarcely containingby-product, 1,3-propanediol, 3-hydroxypropionic acid, acrolein, acrylicacid and acrylic ester can be produced in high yields and with highpurities, respectively. Accordingly, 1,3-propanediol thus produced canbe used as a monomer to be used for producing polyesters andpolyurethanes, and as a starting material for synthesis of cycliccompounds. Further, fibers produced by using this compound do not showdiscoloration because 1,3-propanediol scarcely contains by-product.Also, acrylic acid/acrylic ester thus produced can be used not only forcopolymers for acrylic fibers or for adhesives/agglutinant as anemulsion, but also for coating materials, textile processing, leathers,construction materials, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a gene map of vector 1, which is an expressing plasmidused in Example 1. In FIG. 1A, Amp^(r) represents anampicillin-resistant gene; Cm^(r) represents a chloramphenicol-resistantgene; lacI_(q) represents a lactose repressor gene; and P_(tac)represents a tac promoter, respectively.

FIG. 1B shows a gene map of vector 2, which is an expressing plasmidused in Example 1. In FIG. 1B, Amp^(r) represents anampicillin-resistant gene; Cm^(r) represents a chloramphenicol-resistantgene; lacI_(q) represents a lactose repressor gene; and P_(tac)represents a tac promoter, respectively.

FIG. 1B shows a gene map of vector 1′, which is an expressing plasmidused in Example 2. In FIG. 2A, Amp^(r) represents anampicillin-resistant gene; Cm^(r) represents a chloramphenicol-resistantgene; lacI_(q) represents a lactose repressor gene; and P_(tac)represents a tac promoter, respectively.

FIG. 1B shows a gene map of vector 2′, which is an expressing plasmidused in Example 2. In FIG. 2A, Amp^(r) represents anampicillin-resistant gene; Cm^(r) represents a chloramphenicol-resistantgene; lacI_(q) represents a lactose repressor gene; and P_(tac)represents a tac promoter, respectively.

1. A method for producing 3-hydroxypropionaldehyde which comprises astep of dehydrating glycerin using a microbial cell and/or a treatedmicrobial cell containing diol dehydratase and/or glycerol dehydratase,and optionally diol dehydratase reactivating factor and/or glyceroldehydratase reactivating factor, under conditions so as to give a value(X/Y²) calculated by dividing a catalytic amount [X (U/g glycerin)] ofdiol dehydratase and/or glycerol dehydratase by square of glycerinconcentration [Y (g/100 ml)] within a range of 10 to 8,000, to produce3-hydroxypropionaldehyde.
 2. A method according to claim 1, wherein thedehydration of glycerin is performed using a microbial cell underaerobic conditions.
 3. A method according to claim 1, wherein thedehydration of glycerin is performed using a treated microbial cell. 4.A method for producing 1,3-propanediol which comprises a step ofremoving the microbial cell and/or treated microbial cell from the3-hydroxypropionaldehyde produced by the method set forth in claim 1,subsequently hydrogenating said 3-hydroxypropionaldehyde to produce1,3-propanediol.
 5. A method for producing 3-hydroxypropionic acid whichcomprises a step of oxidizing the 3-hydroxypropionaldehyde produced bythe method set forth in claim 1 to produce 3-hydroxypropionic acid.
 6. Amethod for producing acrolein which comprises a step of reacting the3-hydroxypropionaldehyde produced by the method set forth in claim 1under acidic conditions, to produce acrolein.
 7. A method for producingacrylic acid which comprises a step of oxidizing the acrolein producedby the method set forth in claim 6 to produce acrylic acid.
 8. A methodfor producing an acrylic ester which comprises a step of subjecting theacrolein produced by the method set forth in claim 6 to the oxidativeesterification, to produce an acrylic ester.
 9. A method for producing1,3-propanediol which comprises a step of removing the microbial celland/or treated microbial cell from the 3-hydroxypropionaldehyde producedby the method set forth in claim 2, subsequently hydrogenating said3-hydroxypropionaldehyde to produce 1,3-propanediol.
 10. A method forproducing 1,3-propanediol which comprises a step of removing themicrobial cell and/or treated microbial cell from the3-hydroxypropionaldehyde produced by the method set forth in claim 3,subsequently hydrogenating said 3-hydroxypropionaldehyde to produce1,3-propanediol.
 11. A method for producing 3-hydroxypropionic acidwhich comprises a step of oxidizing the 3-hydroxypropionaldehydeproduced by the method set forth in claim 2 to produce3-hydroxypropionic acid.
 12. A method for producing 3-hydroxypropionicacid which comprises a step of oxidizing the 3-hydroxypropionaldehydeproduced by the method set forth in claim 3 to produce3-hydroxypropionic acid.
 13. A method for producing acrolein whichcomprises a step of reacting the 3-hydroxypropionaldehyde produced bythe method set forth in claim 2 under acidic conditions, to produceacrolein.
 14. A method for producing acrolein which comprises a step ofreacting the 3-hydroxypropionaldehyde produced by the method set forthin claim 3 under acidic conditions, to produce acrolein.
 15. A methodfor producing acrylic acid which comprises a step of oxidizing theacrolein produced by the method set forth in claim 13 to produce acrylicacid.
 16. A method for producing acrylic acid which comprises a step ofoxidizing the acrolein produced by the method set forth in claim 14 toproduce acrylic acid.
 17. A method for producing an acrylic ester whichcomprises a step of subjecting the acrolein produced by the method setforth in claim 13 to the oxidative esterification, to produce an acrylicester.
 18. A method for producing an acrylic ester which comprises astep of subjecting the acrolein produced by the method set forth inclaim 14 to the oxidative esterification, to produce an acrylic ester.